Core binding factor (CBF) was originally identified as a DNA-binding protein that specifically binds to the asymmetric sequence PyGPyGGT, corresponding to the highly conserved "core" site in mammalian type C retrovirus enhancers. CBF binding sites have subsequently been identified in a number of T-cell specific genes, providing evidence for the role of CBF as a T-cell transcription factor. Additional support for CBF's role as a T-cell transcription factor has recently come from knockout mice in which hematopoiesis was found to be blocked at an early stage. Isolation and subsequent cloning of CBF showed the protein to be a heteromer consisting of an ? and ? subunit. The ? subunit contacts the DNA directly, whereas the ? subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by ? in the presence of ?. Binding of the ? subunit to the ? subunit increases the affinity of the ? subunit for the DNA sixfold without altering the sequence specificity. The ? subunit contains a 128 amino acid region displaying a high homology to the Drosophila segmentation protein called Runt. This 128 amino acid domain is referred to as the Runt domain. Glutathione S-Transferase (GST) fusion proteins with the Runt domain alone have shown this domain is responsible for both the DNA-binding and ?-binding capabilities of the ? subunit. Two of the four genes encoding CBF subunits are proto-oncogenes commonly activated in human leukemias. The inversion and translocations identified in these genes are associated with 30% of de novo acute myeloid leukemias in humans. The importance of CBF in leukemia as well as in its normal role as a transcription factor make elucidation of its function at the molecular level extremely interesting and potentially therapeutically useful. In addition, the lack of any resemblance of the Runt domain or CBF? to any known structural motifs makes them important targets for structure determination. The ultimate objective of our work is the structural characterization of the relevant domains of both subunits of CBF using NMR. The aim of this proposal is the complete NMR heteronuclear assignment and structure determination of a Runt domain-DNA complex. The Runt domain construct we have prepared is a 176 amino acid fragment of the ? subunit. This is complexed to an 18 bp DNA duplex to obtain a well-behaved protein-DNA complex. Preliminary 15N-1H HSQC spectra of this complex with 15N-labeled Runt domain showed very low dispersion in the HSQC spectrum which required a 750 MHz instrument to be resolved. Additionally, we have not, to this point, reached concentrations higher than 0.7 mM for this complex, thus also requiring a high field magnet to obtain adequate signal-to-noise. Due to the >30 kDa size of this complex, we have chosen to label the protein with 50% 2H as well as 13C/15N for assignments via triple resonance experiments. This inclusion of 2H should, as has been shown for the trp repressor-DNA complex for example, increase the relevant T2's to values that permit the recording of triple resonance experiments for the assignment process as well as NOESY spectra for structure determination.